Cancer/Testis Antigen 55 is required for cancer cell proliferation and mitochondrial DNA maintenance

Abstract

Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.

Keywords: CT55; Cell proliferation; Mitochondrial DNA; NCI-60.

Figure 1:. CT55 expression is linked to the poor prognosis of patients with cancer.

A) NCI Genomic Data Commons (GDC) analysis showing the survival data of cases with tumors expressing CT55 (585 cases, black line), cases expressing mutated CT55 based on Ensembl Variant Effect Predictor (VEP) impact (71 cases, red line) and cases expressing deleterious mutated CT55 based on a deleterious Sorting Intolerant From Tolerant (SIFT) impact (26 cases, green line). B) Co-expression of CT55 with other CTAs. Among the 561 positively correlated genes with CT55 in NCI-60 cell lines, 51 belong to the CTA database.

Figure 2:. CT55 sustains cell proliferation:

A) Left panels: representative real time images, of UACC-62 control cells (top), or treated with the control (middle) and CT55 (bottom) siRNA for 1, 3 and 5 days, Scale barre = 400 μm. Right panels show the confluence quantifications. B) Upper panels: representative images of crystal violet staining of control and siRNA treated cells after 5 days; and lower panels show the staining quantification (n=6). C) Measurements of the ATP production using the ATPLite assay (n=3). D) Principal component analysis (PCA) of the RNA-seq data shows one cluster including the UACC-62 untreated cells (bleu) and SiCtrl (green) on the right and CT55 silenced cells (red), on the left. E) Total number of genes up (red) or down (blue) regulated with a fold change >2 between control cells (untreated + SiCtrl) and CT55 silenced cells. F) PANTHER gene ontology of enriched pathways including the 1158 downregulated genes. G) RT-qPCR mRNA expression level of PCNA, MKi67 and TOP2A genes involved in cell proliferation. M±SEM (n=3), *: P<0,05; **: P<0,01; ***: P<0,001. Mann-Whitney test.

Figure 3:. CT55 localizes to mitochondria and ER.

Fluorescence microscopy images of UACC-62 cells transfected with the indicated CT55a constructs (as represented at left of each panel) tagged at C-terminal end with a GFP. A, B, C, D and E) Columns from left to right show GFP (green), MitoTracker Red for mitochondrial staining (red), and merged images. A’, B’ and D’) Columns from left to right show GFP (green), DsRed-ER for ER staining (red) and merged images. B and B’) Enlarged views of the inset areas show the co-localization of the green and red staining. Lines drawn in the enlarged views show the relative fluorescence intensity of the red and green staining.

Figure 4:. CT55 controls mtDNA copy number.

A) top panel: mtDNA copy number in HCT-116, SK-MEL-2, SNB-75 and UACC-62 cell lines; bottom panel: Western blot showing the expression level of CT55 isoforms in these four cell lines (n=4). B) Top panel: mtDNA copy number in untreated UACC-62 and after treatment with the control (SiCtrl) and CT55 (SiCT55) siRNAs for 5 days. Bottom panel: Western blot showing the expression level of the two CT55 isoforms before and after siRNA treatment (n=6). C) mtDNA depletion and recovery in UACC-62 cells. Cells were treated with 200 nM of EtBr for 2 days, then released while simultaneously treated with the indicated siRNA in absence of EtBr. mtDNA copy number was quantified at the indicated days (n=4). * P<0,05; ** P<0,01. Test Mann-Whitney (B). Test ANOVA (C). D) mRNA expression levels of CT55 and PDK2 in the same four cell lines. PDK2 mRNA expression is the highest negatively correlated gene to CT55 expression level across NCI-60 database. D) CT55 mRNA expression and mtDNA copy number of untreated and treated HCT-116 cell line with 5-aza-2’-dexoycyditine at 2,5 μM and 5 μM for 3 days. E) Quantification of mtDNA copy number in the NCI-60 cell lines. CellMiner pattern comparison identified CT55 as the second highest positively correlated gene with mtDNA copy number.